J Mol Biol, 1998 Oct 9, 282(5), 959 - 67 Transmembrane helix tilting and ligand-induced conformational changes in the lactose permease determined by site-directed chemical crosslinking in situ; Wu J et al.; The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease of Escherichia coli, each with a Cys residue, were co-expressed independently, and crosslinking was studied . Proximity of paired Cys residues in helices II (position 49, 52, 53, 56, 57, 60, 63 or 67) and VII (position 227, 230, 231, 234, 238, 241, 242 or 245) or XI (position 350, 353, 354, 357, 361 or 364) was examined by using two homobifunctional thiol-specific crosslinking agents of different lengths (6 or 10 A) . The results demonstrate that a Cys residue placed in the periplasmic half of helix II (position 49, 52, 53 or 57) crosslinks to Cys residues in the periplasmic half of helix VII (position 241, 242 or 245) . In contrast, no crosslinking is evident with paired-Cys residues in the cytoplasmic halves of helices II (position 60, 63 or 67) and VII (position 227, 230, 231, 234 or 238) . Remarkably, a Cys residue in the cytoplasmic half of helix II (position 60, 63 or 67) crosslinks with a Cys residue in the cytoplasmic half of helix XI (position 350, 353 or 354), while paired-Cys residues at positions in the periplasmic halves of the two helices do not crosslink . Therefore, helix II is tilted in such a manner that the periplasmic end is close to helix VII, and the cytoplasmic end is close to helix XI . Furthermore, ligand-binding alters the crosslinking efficiency of paired-Cys residues in helices II and VII or XI, indicating that both interfaces are conformationally active . The results are consistent with the conclusion that ligand-binding induces a scissors-like movement of helices II and VII that increases interhelical distance by 3 to 4 A at the periplasmic ends and decreases the distance by 3 to 4 A at the approximate middle of the two transmembrane helices . Biochemistry, 1998 Sep 29, 37(39), 13902 - 9 Importance of central alpha-helices of human apolipoprotein A-I in the maturation of high-density lipoproteins; Frank PG et al.; We have studied the role of amphipathic alpha-helices in the ability of apoA-I to promote cholesterol efflux from human skin fibroblasts and activate lecithin:cholesterol acyltransferase (LCAT) . Three apoA-I mutants were designed, each by deletion of a pair of predicted adjacent central alpha-helices {Delta(100-143), Delta(122-165), Delta(144-186)}, and expressed in Escherichia coli . This strategy was used to minimize disruption of the predicted secondary structure of the resulting protein . These three central deletion mutants have been previously shown to be expressed as stable folded proteins but to exhibit altered phospholipid-binding properties . When recombined with phospholipids to form homogeneous LpA-I containing equivalent amounts of POPC and tested for their ability to promote diffusional cholesterol efflux from normal {3H}cholesterol-labeled fibroblasts, each mutant and the wild-type recombinant protein (Rec.-apoA-I) promoted cholesterol efflux with very similar rates at all the concentrations tested . These experiments showed that all LpA-I could acquire cellular cholesterol with similar affinity and binding capacity . However, when the cell-incubated LpA-I were incubated with purified LCAT, two mutants, Delta(122-165) and Delta(144-186), appeared incapable of activating the enzyme . To directly determine their ability to activate LCAT, each mutant and the control were recombined with equivalent amounts of cholesterol and phospholipid and incubated with the purified enzyme . The results show that whereas deletion of residues 100-143 has little effect on LCAT activation, deletion of residues 122-165 or 144-186 results in an inability of the mutants to promote cholesterol esterification . In conclusion, our results show that no specific sequence in the central domain of apoA-I is required for efficient diffusional cholesterol efflux from normal fibroblasts; however, residues 144-186 appear critical for optimum LCAT activation and cholesteryl ester accumulation . Since deletion of residues 144-186 also perturbs phospholipid association and prevents the formation of large LpA-I particles {Frank, P . G., Bergeron, J., Emmanuel, F., Lavigne, J . P., Sparks, D . L., Denefle, P., Rassart, E., and Marcel, Y . L . (1997) Biochemistry 36, 1798-1806}, the data show that this pair of alpha-helices plays an important role in the maturation of HDL . Sequence analysis of these apoA-I helices further identifies specific residues that appear essential to this activity. Biochemistry, 1998 Sep 29, 37(39), 13893 - 901 D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine; Sage CR et al.; In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate . All mutants of D221 except cysteine abolish activity . We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N . In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual . CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes . The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form . In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites . In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor . In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor . This orientation blocks the conformational change required for forming covalent ternary complexes . Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring . Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis. Biochemistry, 1998 Sep 29, 37(39), 13871 - 81 The oligomycin sensitivity conferring protein of rat liver mitochondrial ATP synthase: arginine 94 is important for the binding of OSCP to F1; Golden TR et al.; The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis . To gain insight into the function of OSCP, mutations were made in a highly conserved central region of the subunit, and the recombinant proteins were studied using several biochemical assays . Rat liver OSCP was expressed to high levels in Escherichia coli, solubilized from inclusion bodies, renatured, and purified to homogeneity . The recombinant protein was able to reconstitute oligomycin-sensitive ATPase activity to inner membrane vesicles depleted of F1 and OSCP, and bound to F1 with a stoichiometry of 1:1 . A novel fluorescence anisotropy assay was developed to study the affinity of binding of F1 to OSCP, providing a Kd value of 51 +/- 11 nM . Two highly conserved, charged residues (E91 and R94) which lie within the central region of OSCP were mutated, and the recombinant proteins (E91Q, R94Q, and R94A) were purified to homogeneity and judged by CD spectroscopy to have structures similar to that of the wild-type protein . Both R94 mutants demonstrated little or no binding to F1, while the E91Q bound in a manner identical to that of wild-type OSCP . Significantly, all three mutant proteins were able to reconstitute F1 with membranes and to confer oligomycin sensitivity to the same extent as wild-type OSCP . These results demonstrate that a single tight binding site exists on isolated rat liver F1 for OSCP, and implicate arginine 94 as playing a critical role in this site . In addition, these results indicate that this tight binding site is not required for conferral of oligomycin sensitivity to the reconstituted F0F1 complex. Biochemistry, 1998 Sep 29, 37(39), 13800 - 6 A conserved aspartate residue, Asp187, is important for Na+-dependent proline binding and transport by the Na+/proline transporter of Escherichia coli; Quick M et al.; Asp187 in the Na+/proline transporter of Escherichia coli (PutP) is conserved within the Na+/solute cotransporter family . Information on the role of this residue has been gained by amino acid substitution analysis . PutP with Glu, Asn, or Cys in place of Asp187 catalyzed Na+-coupled proline uptake at 75%, 25%, and 1.5%, respectively, of the Vmax of PutP-wild-type while the apparent Km for proline was only slightly altered . Importantly, acetylation or amidoacetylation of an engineered transporter containing a single Cys at position 187 stimulated proline uptake . Strikingly, PutP-D187C exhibited high-affinity proline binding even at very low Na+ concentrations (2 microM) while proline binding to PutP-wild-type, -D187E, and -D187N was strictly dependent on the Na+ concentration . The apparent independence of proline binding from the Na+ concentration can at least partially be attributed to an enhanced Na+ affinity of PutP-D187C . In addition, reaction of PutP containing a single Cys at position 187 with N-ethylmaleimide was inhibited by Na+ but not by Li+ or proline . The results indicate that electrostatic interactions of the amino acid side chain at position 187 in PutP with other parts of the transporter and/or the coupling ion are crucial for active proline transport . It is suggested that Asp187 is located close to the pathway of the coupling ion through the membrane and may be involved in the release of Na+ on the cytoplasmic side of the membrane. Biochemistry, 1998 Sep 29, 37(39), 13736 - 43 Effects of secondary structure on DNA and RNA cleavage by diplatinum(II); Carter PJ et al.; The photochemistry of Pt2(pop)44- with nucleic acids has been studied using radiolabeled oligomers of DNA and RNA and high-resolution electrophoresis (pop is P2O5H22-) . Photolysis of Pt2(pop)44- with duplex DNA produces an even cleavage ladder and relatively little enhancement of cleavage upon treatment with piperidine . In contrast, the cleavage pattern is far less regular with single-stranded DNA, and there is a large enhancement in cleavage upon treatment with piperidine . Accordingly, photolysis of Pt2(pop)44- with the DNA hairpin 5'-d{ATCCTATTTATAGGAT} produces a much larger piperidine enhancement at the loop and end nucleotides than in the stem . There is an additional piperidine enhancement that occurs selectively at guanine residues either in RNA or in DNA at low Mg2+ concentrations that is attributed to outer-sphere electron transfer on the basis of the known excited-state redox potentials of Pt2(pop)44- and the expected oxidative chemistry of guanine . The extent of guanine oxidation is higher compared to the extent of sugar oxidation at low Mg2+ concentrations, which can be attributed to a shallower distance dependence for electron transfer compared to that for atom transfer . The effects of Mg2+ and piperidine or aniline treatment were examined on stem-loop structures of DNA and RNA and gave partial images of the expected secondary structures. Biochemistry, 1998 Sep 29, 37(39), 13710 - 9 The human immunodeficiency virus type 1 Vpu protein enhances membrane permeability; Gonzalez ME et al.; Infection of T lymphocytes by the human immunodeficiency virus causes drastic alterations in the intracellular cation content of the infected cells . The human immunodeficiency virus type 1 genome encodes several accessory proteins, including Vpu, an integral membrane protein that forms ion channels in planar lipid bilayers . The effect of Vpu on the permeability of the plasma membrane to several molecules has been analyzed . Expression of vpu in Escherichia coli cells increases membrane permeability to a number of molecules such as 2-nitrophenyl beta-D-galactopyranoside, uridine, the impermeable translation inhibitor hygromycin B, and lysozyme . In addition, transient expression of Vpu in eukaryotic COS cells enhances entry of charged molecules such as hygromycin B and neurobiotin into these cells . The effect of Vpu on cell membrane permeability resembles that reported for other membrane-active proteins from different animal viruses, including influenza M2, Semliki Forest virus 6K, and poliovirus 2B and 3A proteins. Biochemistry, 1998 Sep 29, 37(39), 13604 - 13 Lys75 of Anabaena ferredoxin-NADP+ reductase is a critical residue for binding ferredoxin and flavodoxin during electron transfer; Martinez-Julvez M et al.; Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in the interaction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld) . To assess this possibility, this residue has been replaced by another positively charged residue, Arg, by two uncharged residues, Gln and Ser, and by a negatively charged residue, Glu . UV-vis absorption, fluorescence, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate that all the mutated proteins folded properly and that significant protein structural rearrangements did not occur . Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP, indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) between FNR and NADP+ or NADPH . However, steady-state kinetic activities requiring complex formation and ET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75 was removed, and the ET reaction was not even measurable if a negatively charged residue was placed at this position . These kinetic parameters also suggest that it is complex formation that is affected by mutation . Consistent with this, when dissociation constants (Kd) for FNRox-Fdox (differential spectroscopy) and FNRox-Fdrd (laser flash photolysis) were measured, it was found that neutralization of the positive charge at position 75 increased the Kd values by 50-100-fold, and that no complex formation could be detected upon introduction of a negative charge at this position . Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects the complex formation process with its protein partners but indicates that ET is still achieved in all the reactions . This study thus clearly establishes the requirement of a positive charge at position Lys75 for complex formation during ET between FNR and its physiological protein partners . The results also suggest that the interaction of this residue with its protein partners is not structurally specific, since Lys75 can still be efficiently substituted by an arginine, but is definitely charge specific. Biochemistry, 1998 Sep 29, 37(39), 13499 - 506 The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate; Reczkowski RS et al.; S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L-methionine and the subsequent hydrolysis of the PPPi to PPi and Pi before product release . Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain . Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site . Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif . Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p-hydroxyphenylglyoxal inactivates the enzyme . ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site . Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H) . In the overall reaction, the R244L mutant has the kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat . In contrast, the kcat values for hydrolysis of added PPPi by the R244L and R244H mutants have been reduced by less than 2 orders of magnitude . In contrast to the wild-type enzyme in which 98% of the Pi formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of Pi from the alpha- and gamma-phosphoryl groups of ATP . Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction. Transplantation, 1998 Sep 15, 66(5), 567 - 72 Alterations in mRNA for inducible and endothelial nitric oxide synthase and plasma nitric oxide with rejection and/or infection of allotransplanted lungs; Wang X et al.; BACKGROUND: Experiments were designed to determine expression of type II (iNOS) and type III (ecNOS) nitric oxide synthase in lung parenchyma and systemic endothelial cells with rejection and/or infection of single lung allografts . METHODS: After single lung allotransplantation, dogs were maintained on standard triple immunosuppressive therapy for 5 days and then placed into one of three groups . Group I (n=4) was maintained on immunosuppressants, group II (n=7) immunosuppression was withdrawn to allow acute rejection of the allograft, and group III (n=6) infection was induced by bronchoscopic inoculation of Escherichia coli . RESULTS: At postoperative days 7-9, no histological evidence of rejection or infection was observed in transplanted lungs of group I . In lungs of group II, rejection ranged from mild to severe; in lungs of group III, infection was severe . Some animals had both rejection and infection (n=8) and were studied separately . Plasma levels of nitric oxide increased comparably with rejection and/or infection compared to preoperative values . Expression of mRNA for ecNOS decreased significantly in lung parenchyma but not in aortic endothelial cells from dogs of groups II and III . However, expression of mRNA for iNOS increased with both rejection and/or infection in both lung parenchyma and aortic endothelial cells . CONCLUSIONS: iNOS is induced locally within the graft and systemically in aortic endothelial cells with rejection and/or infection of lung allografts . Plasma levels of nitric oxide are elevated with both rejection and infection and may not be useful in the differential diagnosis of these processes after lung transplantation. Cell, 1998 Sep 18, 94(6), 829 - 39 Structure of type IIbeta phosphatidylinositol phosphate kinase: a protein kinase fold flattened for interfacial phosphorylation; Rao VD et al.; Phosphoinositide kinases play central roles in signal transduction by phosphorylating the inositol ring at specific positions . The structure of one such enzyme, type IIbeta phosphatidylinositol phosphate kinase, reveals a protein kinase ATP-binding core and demonstrates that all phosphoinositide kinases belong to one superfamily . The enzyme is a disc-shaped homodimer with a 33 x 48 A basic flat face that suggests an electrostatic mechanism for plasma membrane targeting . Conserved basic residues form a putative phosphatidylinositol phosphate specificity site . The substrate-binding site is open on one side, consistent with dual specificity for phosphatidylinositol 3- and 5-phosphates . A modeled complex with membrane-bound substrate and ATP shows how a phosphoinositide kinase can phosphorylate its substrate in situ at the membrane interface. Dig Dis Sci, 1998 Sep, 43(9 Suppl), 160S - 166S Effects of rebamipide on production of several cytokines by human peripheral blood mononuclear cells; Aihara M et al.; Recently, the relative contributions of local T helper cell responses of the Th1-type and Th2-type to the pathogenesis of gastritis and peptic ulcers associated with Helicobacter pylori infection have been examined . However, the results were controversial with respect to whether cellular immunity (Th1-type) or humoral immunity (Th2-type) responses predominate in H . pylori infection and with respect to how these responses may contribute to disease pathogenesis . In this study, we investigated the characteristics of the production of various cytokines induced by H . pylori or lipopolysaccharide (LPS), which was derived from H . pylori or Escherichia coli, in human peripheral blood mononuclear cells (PBMC) . Live H . pylori induced production of many cytokines, such as IL-1beta, IL-10, IL-8, IFN-gamma, and TNF-alpha, whereas we could not detect IL-2 or IL-4 . Moreover, we evaluated the effect of rebamipide on the production of several cytokines from PBMC induced by various stimuli . Rebamipide suppressed the production of IL-8, IL-10, TNF-alpha, and IL-1beta induced by H . pylori in a dose-dependent manner . On the other hand, the production of IL-12 induced by H . pylori showed a tendency to increase as a result of treatment of the cells with rebamipide . These results suggested that rebamipide might be effective in regulating cytokine responses in the H . pylori-infected host and maintaining host immunity . Moreover, it might contribute positively to disease progression and bacterial eradication. Dig Dis Sci, 1998 Sep, 43(9 Suppl), 154S - 159S Effect of rebamipide on liver damage and increased tumor necrosis factor in a rat model of endotoxin shock; Hong KW et al.; We investigated the effect of rebamipide, a novel antiinflammatory agent, on liver damage in a rat model of circulatory shock induced by bacterial endotoxin (E . coli lipopolysaccharide, LPS) . Endotoxemia for 6 hr resulted in a 5.9-fold rise in the serum levels of nitrite (P < 0.05) with a significant rise in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactic dehydrogenase (LDH), suggestive of liver dysfunction . The increased activities of serum ALT, AST, and LDH, but not serum nitrite were significantly inhibited by rebamipide (100 mg/kg, orally for five days) . Myeloperoxidase activity in the liver was significantly elevated in the rats with endotoxemia by 2.4-fold (P < 0.05), which was also significantly inhibited by rebamipide . Upon LPS injection, serum TNF-alpha levels peaked at 1 hr after LPS (from 167.4 +/- 20.0 to 1570.0 +/- 100.0 pg/ml) and thereafter rapidly declined . The increased TNF-alpha level measured at 1 hr was significantly inhibited by pretreatment with rebamipide (100 mg/kg for five days) . It is suggested that rebamipide exerts a strong protective effect on the LPS-induced liver damage through inhibition of activation of neutrophils and TNF-alpha production. Eur J Neurosci, 1998 Mar, 10(3), 989 - 99 Regulation of cell-type specific expression of lacZ by the 5'-flanking region of mouse GAD67 gene in the central nervous system of transgenic mice; Katarova Z et al.; The transcriptional regulation of the murine gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was studied by beta-galactosidase histochemistry in transgenic mice carrying fusion genes between progressively longer portions of the 5'-upstream regulatory region of GAD67 and E . coli lacZ . No expression was detected in brains of mice carrying 1.3 kb of upstream sequences including a housekeeping and two conventional promoters, and two negative regulatory elements with homology to known silencers . In mice carrying the same portion of the promoter region plus the first intron, lacZ expression in the adult central nervous system was found in few, exclusively neuronal sites . The number of correctly stained GABAergic centres increased dramatically with increasing the length of the 5'-upstream region included in the construct which suggests that multiple putative spatial enhancers are located in this region . Their action is influenced by epigenetic mechanisms that may be due to site-of-integration and transgene copy-number effects . Additional cis-acting elements are needed to obtain fully correct expression in all GABAergic neurons of the adult central nervous system. FEMS Immunol Med Microbiol, 1998 Aug, 21(4), 313 - 21 In vitro inhibition of adhesion of enterotoxigenic Escherichia coli K88 to piglet intestinal mucus by egg-yolk antibodies; Jin LZ et al.; The objective of the study was to determine if the adhesion of E . coli K88 to piglet intestinal mucus could be inhibited in vitro by spray-dried egg-yolk anti-K88 antibodies . Binding of E . coli was monitored using a radioactive assay . Four 14+/-2-day-old healthy piglets were used for the preparation of mucus from the small intestine . Competition and displacement phenomena were investigated by incubating (a) egg-yolk antibodies and E . coli together prior to adding to the mucus and (b) E . coli and mucus, followed by egg-yolk antibodies . The results demonstrated that egg-yolk antibodies inhibited the adhesion of 3H-labeled local strain of hemolytic E . coli K88+ (E . coli K88+ MB) to piglet small intestinal mucus by 84.6-97.0% when the egg-yolk antibodies were diluted 10, 20, 40 or 100 times . The adhesion inhibiting effects of egg-yolk antibodies declined dramatically when the antibody dilution was more than 250-fold . A similar adhesion inhibiting effect was observed when egg-yolk antibodies were incubated with E . coli K88+ MB for 15, 30 and 60 min prior to the adhesion test . Egg-yolk antibodies when diluted 50- and 100-fold had a very strong inhibiting ability against E . coli K88+ MB at a concentration of 10(9) colony forming units (cfu) ml(-1) (adhesion was < 6%) . However, dilution of 100 times for egg yolk antibodies was insufficient to inhibit the adhesion of E . coli K88+ MB to intestinal mucus when the concentration of E . coli K88+ MB was 10(10) cfu ml(-1) . The displacement test indicated that there was no significant reduction in the adhesion of E . coli K88+ MB to the small intestinal mucus when egg-yolk antibodies were added after adhesion of the organism to the mucus . These studies demonstrate that anti-K88+ MB fimbriae antibodies from chicken egg-yolk when added to E . coli K88+ MB prevented their binding to receptors in the mucus isolated from the intestine of piglets. Chirurgie, 1998 Apr, 123(2), 168 - 74 {Gene therapy of cerebral glioblastoma by adenovirus vector . Experimental model in the rat}; Dufour T et al.; Our aim was to test the therapeutic effects of adenovirus-mediated gene therapy in an animal brain tumor model which was obtained by stereotactic injection of 9L gliosarcoma cells into the caudate nucleus of rat brains . Seven days after the implantation of tumor cells, adenovirus vectors bearing the Escherichia coli beta-galactosidase gene (ADVbgal) or the herpes simplex virus thymidine kinase gene (ADVtk) were stereotactically injected into the tumor . Injection of the ADVbgal resulted in the expression of the marker gene in 11 animals . Transfer of the ADVtk was followed, 3 days later, by intraperitoneal injection of ganciclovir (GCV) for 10 days . A control group was treated with saline instead of GCV . We observed a significant regression of the tumors in the rats treated with ADVtk and GCV as compared with control animals . In four cases the tumor completely disappeared after treatment . These results demonstrate the potential efficacy of adenovirus-mediated transfer of the HSVtk gene following by GCV administration for the treatment of glioblastomas. J Biomol NMR, 1998 Aug, 12(2), 319 - 24 Rapid measurement of scalar three-bond 1HN-1H alpha spin coupling constants in 15N-labelled proteins; Ponstingl H et al.; Two new 2D NMR experiments, CT-HMQC-HA and CT-HMQC-HN, are proposed for the rapid measurement of homonuclear 3JHNH alpha coupling constants of uniformly 15N-enriched proteins in solution . The experiments are based on the comparison of the signal intensities in a pair of constant-time {15N,1H}-HMQC spectra recorded with and without decoupling of the amide proton-alpha proton coupling . Experimental data recorded with the 78-residue N-terminal domain of the E . coli arginine repressor (ArgR-N) and with oxidized E . coli flavodoxin (176 residues) showed good agreement with 3JHNH alpha coupling constants obtained by fitting of the multiplet fine structure of the amide proton resonances or from a 3D HNHA-J experiment, respectively . Quantitative estimates for the effects from different relaxation rates of in-phase and antiphase magnetization are given. J Biomol NMR, 1998 Aug, 12(2), 299 - 306 An asymmetric deuterium labeling strategy to identify interprotomer and intraprotomer NOEs in oligomeric proteins; Jasanoff A; A major difficulty in determining the structure of an oligomeric protein by NMR is the problem of distinguishing inter- from intraprotomer NOEs . In order to address this issue in studies of the 27 kD compact trimeric domain of the MHC class II-associated invariant chain, we compared the 13C NOESY-HSQC spectrum of a uniformly 13C-labeled trimer with the spectrum of the same trimer labeled with 13C in only one protomer, and with deuterium in the other two protomers . The spectrum of the unmixed trimer included both inter- and intraprotomer NOEs while the spectrum of the mixed trimer included only intraprotomer peaks . NOEs clearly absent from the spectrum of the mixed trimer could be confidently assigned to interprotomer interactions . Asymmetrically labeled trimers were isolated by refolding a 13C-labeled shorter form of the protein with a 2H-labeled longer form, chromatographically purifying trimers with only one short chain, and then processing the trypsin to yield only protomers with the desired N- and C-termini . In contrast to earlier studies, in which statistical mixtures of differently labeled protomers were analyzed, our procedure generated only a well-defined 1:2 oligomer, and no other mixed oligomers were present . This increased the maximum possible concentration of NMR-active protomers and thus the sensitivity of the experiments . Related methods should be applicable to many oligomeric proteins, particularly those with slow protomer exchange rates. J Biomol NMR, 1998 Aug, 12(2), 259 - 76 Some NMR experiments and a structure determination employing a {15N,2H} enriched protein; Mal TK et al.; We present the results of studies of an aqueous sample of a highly {15N,2H} enriched protein, the SH3 domain from Fyn . Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances . The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration . Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances . The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7A apart . A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments . These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR . These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain. J Biomol NMR, 1998 Aug, 12(2), 209 - 22 Hydrogen bonding geometry of a protein-bound carbohydrate from water exchange-mediated cross-relaxation; Sayers EW et al.; We present heteronuclear two-dimensional methods for the analysis of the geometry of exchangeable protons on a protein-bound carbohydrate . By using a water-selective NOESY-HSQC, we observed cross-relaxation between carbohydrate hydroxyl protons and non-exchangeable ring protons in the complex of {13C6}-alpha-methyl-D-mannopyranoside with recombinant rat mannose binding protein . Using a simple kinetic model, we were able to explain the differences in the initial slopes of the resulting cross-relaxation buildup curves in terms of the geometry of the hydroxyl protons in the bound state . The hydroxyl rotamers consistent with our cross-relaxation data fit very well with predictions based on the crystal structure of MBP bound to a mannose-rich oligosaccharide . These methods should be applicable to other systems where both ligand exchange and water exchange are fast relative to the rate of cross-relaxation. Gene, 1998 Sep 18, 218(1-2), 57 - 61 A new set of positive/negative selectable markers for mammalian cells; Karreman C; Five new positive and negative selectable markers were created for use in mammalian cells . Their negative selectabilities are based on the Thymidine kinase (Tk) gene of Herpes Simplex virus (HSV) or the Cytidine deaminase (codA) gene of E . coli . The markers can be selected positively by their ability to induce either Hygromycin (Hyg), neomycin (neo), puromycin (PAC) or Blasticidin S (BlaS) resistance . With these markers, two complete sets of markergenes are available that induce independent negative selectable phenotypes. Gene, 1998 Sep 18, 218(1-2), 49 - 56 Sequence and activity of parathyroid hormone/parathyroid hormone-related protein receptor promoter region in human osteoblast-like cells; Manen D et al.; The parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor gene has been characterized in various species . The structure of its promoter and the regulation of its expression in human tissues have, however, not been clearly established yet . We characterized the region upstream of the PTH/PTHrP receptor gene and investigated its promoter activity in the human osteoblast-like SaOS-2 cells . In this region, three untranslated exons were localized, U1 and U2 by using a kidney cDNA, and U3 by homology with the mouse gene . In human osteoblast-like cells, a distal promoter (P1) was found to be inactive, as evaluated by luciferase reporter gene assays, in contrast to the situation found in human and mouse kidney tissue . A second promoter (P2), previously described in mouse and human kidney, was shown to be active in human osteoblast-like SaOS-2 cells . We found a hitherto uncharacterized promoter (P3), closely upstream of the ATG start codon . The activities of P2 and P3 were not additive . These results provide important information on the structure of the 5' flanking region of the human PTH/PTHrP gene receptor. Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11897 - 902 Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy; Elefanty AG et al.; Gene targeting experiments have demonstrated that the transcription factor SCL is essential for primitive and definitive hematopoiesis in the mouse . To study the functional properties of hematopoietic cells expressing SCL, we have generated mutant mice (SCLlacZ/w) in which the Escherichia coli lacZ reporter gene has been "knocked in" to the SCL locus, thereby linking beta-galactosidase expression to transcription from the SCL promoter . Bone marrow cells from heterozygous SCLlacZ/w mice were sorted into fractions expressing high, intermediate and low levels of beta-galactosidase (designated lacZhigh, lacZint, and lacZneg) . Cells that were lacZhigh or lacZint were enriched for day 12 spleen colony-forming units and myeloid and erythroid colony-forming cells (CFCs) . These fractions included >99% of the erythroid and >90% of the myeloid CFCs . Culture of sorted bone marrow populations on stromal cells secreting interleukin-7 or in fetal thymic organ cultures showed that B and T lymphoid progenitors were also present in the lacZhigh and lacZint fractions . These data provide a functional correlation between SCL expression and colony-forming ability in immature hematopoietic cells . Our data also suggested that expression of SCL was transient and confined to hematopoietic stem and/or progenitor cells, because the differentiated progeny of most lineages (except the erythroid) were beta-galactosidase-negative. Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11643 - 8 Thermodynamics of electron transfer in Escherichia coli cytochrome bo3; Schultz BE et al.; The proton translocation mechanism of the Escherichia coli cytochrome bo3 complex is intimately tied to the electron transfers within the enzyme . Herein we evaluate two models of proton translocation in this enzyme, a cytochrome c oxidase-type ion-pump and a Q-cycle mechanism, on the basis of the thermodynamics of electron transfer . We conclude that from a thermodynamic standpoint, a Q-cycle is the more favorable mechanism for proton translocation and is likely occurring in the enzyme. Nature, 1998 Sep 17, 395(6699), 244 - 50 Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d; Brotherton DH et al.; The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19 INK4d protein has been determined at 1.9 A resolution . The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind . The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation . The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs . Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer. Nature, 1998 Sep 17, 395(6699), 237 - 43 Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a; Russo AA et al.; The cyclin-dependent kinases 4 and 6 (Cdk4/6) that control the G1 phase of the cell cycle and their inhibitor, the p16INK4a tumour suppressor, have a central role in cell proliferation and in tumorigenesis . The structures of Cdk6 bound to p16INK4a and to the related p19INK4d reveal that the INK4 inhibitors bind next to the ATP-binding site of the catalytic cleft, opposite where the activating cyclin subunit binds . They prevent cyclin binding indirectly by causing structural changes that propagate to the cyclin-binding site . The INK4 inhibitors also distort the kinase catalytic cleft and interfere with ATP binding, which explains how they can inhibit the preassembled Cdk4/6-cyclin D complexes as well . Tumour-derived mutations in INK4a and Cdk4 map to interface contacts, solidifying the role of CDK binding and inhibition in the tumour suppressor activity of p16INK4a. Cancer Lett, 1998 Aug 14, 130(1-2), 127 - 31 Stannous chloride and the glucoheptonic acid effect: study of a kit used in nuclear medicine; Assis ML et al.; Stannous dichloride is used as a reducing agent in the preparation of technetium-99m radiopharmaceuticals . We decided to evaluate the genotoxic potential of the tin (II)-glucoheptonate complex in the kit using a tester strain of Escherichia coli AB1157 . Our results show that tin (II) chloride and the tin (II)-glucoheptonate complex exert a genotoxic effect in this system . While the genotoxic effect disappeared when the glucoheptonate concentration was increased, the glucoheptonate did not protect the cultures from the damaging effects of hydrogen peroxide . The ability of glucoheptonate to protect cultures from tin (II)-induced damage can be explained on the basis of its metal chelating properties. J Neurochem, 1998 Oct, 71(4), 1369 - 80 Cloning and characterization of a novel form of tyrosine hydroxylase from the human parasite, Schistosoma mansoni; Hamdan FF et al.; Catecholamines such as dopamine and noradrenaline play important roles as neuromuscular transmitters and modulators in all parasitic helminths, including the human parasite, Schistosoma mansoni . We have cloned a novel S . mansoni tyrosine hydroxylase (SmTH) cDNA that shows high homology to mammalian tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines . Two subsets of SmTH transcripts were identified, one of which carries the S . mansoni spliced-leader (SL) sequence at its 5' end, whereas the other does not appear to be trans-spliced to the S . mansoni SL . The two types of SmTH transcripts encode the same protein of 465 amino acids and a predicted size of 54 kDa . Expression of SmTH as an N-terminal histidine fusion protein in Escherichia coli produced an active enzyme that was purified approximately 52-fold to apparent homogeneity and had a final specific activity of 0.78 micromol/min/mg . The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and the same sensitivity to inhibition by high concentrations of the substrate, tyrosine, as the mammalian enzyme . Purified SmTH also showed characteristic inhibition by catecholamine products, although the sensitivity to product inhibition was lower than that of the mammalian enzyme . This evidence indicates that SmTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian host's enzyme but may differ in its properties of regulation . This first demonstration of tyrosine hydroxylase in a parasitic helminth further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously. Methods Enzymol, 1998, 295, 424 - 50 Energetic methods to study bifunctional biotin operon repressor; Beckett D; Application of a broad range of approaches and techniques to analysis of the functional energetics of the biotin regulatory system has enabled dissection of each of the steps in the assembly of this transcriptional repression complex . Although the molecular details of the interactions are not yet completely understood, the studies described in this article have laid a solid foundation for future studies of the system . The application of kinetic and equilibrium methods to studies of binding of the allosteric effector has allow determination of the kinetic parameters governing the interaction of the protein and ligand . The kinetic parameters have, furthermore, been utilized to calculate the equilibrium parameters associated with the binding . The great advantage of using kinetic methods to study the binding process is the additional information provide about the mechanism of allosteric activation of the protein . Based on the initial observation of a kinetic time course that is consistent with the occurrence of a structural change concomitant with effector binding, additional measurements have been performed that have allowed formulation of a testable hypothesis concerning the nature and location of one locus: the structural change in the three-dimensional structure of BirA . Studies of assembly of the protein indicate the bio-5-AMP is an allosteric activator of dimerization of the protein . The dimerization is, however, weak . These results have been critical in analyzing site-specific DNA binding measurements . Application of the DNase I footprinting technique has allowed formulation of a model for association of holoBirA with bioO . Results of studies of binding of the protein to mutant operator templates, although not yielding the anticipated results, provide further insight into the mechanism of association of the protein and DNA . Two models for binding, the validity of which can be tested via the application of kinetic techniques, have been derived from these measurements . The results of quantitative studies of the biotin regulatory system can be interpreted in the context of the biological function of the system . The biotin holoenzyme ligases are a class of enzymes found across the evolutionary spectrum . Only a subset of these enzymes, including BirA, also function as transcriptional repressors . The tight binding of the allosteric effector may be understood in light of the bifunctional nature of the BirA-bio-5'-AMP complex . It is possible that the unusually high thermodynamic and kinetic stability of the complex ensures that the most probable state of the protein in vivo is the adenylate-bound form . This complex, not the unliganded protein, is active in both enzymatic transfer of biotin and site-specific DNA binding . This ensures that on depletion of the intracellular pool of apoBCCP, BirA-bio-5'-AMP accumulates and binds to bioO to repress transcription of the biotin biosynthesis operon . The intracellular demand for and synthesis of biotin are, consequently, tightly coupled in the system . The dimerization that accompanies adenylate binding to BirA appears to be significant for site-specific binding of the protein to bioO . Functionally, the simultaneous binding of the two monomers to the two operator half-sites, regardless of the kinetic mechanism by which it occurs, ensures coordinate regulation of transcription initiation from both biotin operon promoters . The multifaceted approach utilized in studies of the biotin regulatory system can serve as a model for studies of any complex transcriptional regulatory system . It is critical in elucidating the functional energetics of any of these systems that the assembly first be dissected into the constituent interactions and that each of these interactions be studied in isolation . This is not only critical for understanding the physicochemical properties of each individual contributing interaction, but is also a necessary precursor to studies of thermodynamic linkage in the system . (AB Arch Biochem Biophys, 1998 Oct 1, 358(1), 182 - 8 The N-terminal region is important for the allosteric activation and inhibition of the Escherichia coli ADP-glucose pyrophosphorylase; Wu MX et al.; The ADPglucose pyrophosphorylase (EC 2.7.7.27) from Escherichia coli is allosterically activated by fructose 1,6-bisphosphate and inhibited by AMP . Proteolysis of the enzyme with proteinase K causes loss of activity and generates two peptides, 21 and 28 kDa, from the 49.7-kDa subunit . The presence of ADPglucose, Mg2+, and fructose 1, 6-bisphosphate during the incubation with proteinase K protected the enzyme activity and prevented cleavage at sites Met181-Ala182 and Phe192-Val193 . Proteolysis of the protected enzyme removed 10 to 13 amino acids from the N-terminal and 2 amino acids from the C-terminal . The resulting enzyme was almost independent of the need for fructose 1,6-bisphosphate for maximal activity and insensitive to inhibition by AMP . The apparent affinity for the substrates was similar to that of the fully-activated wild-type enzyme . These data suggest that amino acid residues in the N-terminal portion and possibly the C-terminal portion of ADPglucose pyrophosphorylase are part of the regulatory domain of the enzyme, critical for allosteric regulation of the enzyme . Arch Biochem Biophys, 1998 Oct 1, 358(1), 104 - 15 Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase; Shiraishi N et al.; The cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (EC 1.6.6.3) was overexpressed in Escherichia coli with a His-tag for purification after mutation of the NADPH binding site . The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochrome b reductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1) . Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase of N . crassa) greatly increased preference for NADH . This mutant had nearly the same NADH:ferricyanide reductase kcat as wild-type with NADPH . Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did . The cytochrome b reductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site . Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH . Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17 . Thus, the specificity ratio was reversed by over 6000-fold by a single mutation . Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2' phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2) . Anal Biochem, 1998 Oct 1, 263(1), 51 - 6 A continuous fluorimetric assay for tail-specific protease; Beebe KD et al.; A continuous fluorimetric assay for tail-specific protease (Tsp) has been developed using a fluorescence donor/quencher system, in which 5-{(2-aminoethyl) amino}naphthalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) are attached to the N-terminus and the lysyl side chain of peptide AARAAK-(6-aminocaproyl)2-ENYALAA, respectively . Tsp-mediated cleavage of the Ala-Arg peptide bond separates the quencher, DABCYL, from the donor, EDANS, and results in a large increase in the fluorescent yield of EDANS (>50-fold) . Using this sensitive assay, Escherichia coli tail-specific protease was shown to exhibit typical Michaelis-Menten kinetics with a kcat of 0.086 +/- 0.002 s-1, KM of 4.0 +/- 0.3 microM, and kcat/KM of 2.2 x 10(4) M-1 s-1 . A control substrate, which only differs from the above substrate by having a charged residue (glutamate) at the C-terminus, showed drastically reduced activity to Tsp (kcat/KM = 58 M-1 s-1) . A peptide containing the C-terminal sequence of the substrate, GRGYALAA, was shown to be a competitive inhibitor of Tsp with a KI value of 31 microM . These results demonstrate the utility of this assay for the rapid assessment of Tsp activity . Anal Biochem, 1998 Sep 10, 262(2), 122 - 8 Site-specific, enzymatic biotinylation of recombinant proteins in Spodoptera frugiperda cells using biotin acceptor peptides; Duffy S et al.; Site-specific, enzymatic biotinylation of recombinant proteins can be exploited to circumvent many problems associated with the use of biotinylating reagents in vitro and to overcome some of their inherent limitations . Additionally, biotinyl proteins can be purified to near-homogeneity in a single step under native conditions . Here we report that a biotin acceptor peptide (BAP) substrate for Escherichia coli biotin holoenzyme synthetase (BirA) can be used to label recombinant proteins with biotin in Spodoptera frugiperda (Sf9) cells, and we describe a collection of baculovirus transfer vectors specifically designed for this purpose . These BioBac vectors will greatly expand the range of proteins to which this technology can be applied . Anal Biochem, 1998 Sep 10, 262(2), 110 - 21 Identification of biotinylation sites on proteins by selective retrieval of 2-iminobiotinylated peptides from proteolytic peptide mixtures: localization of the accessible lysine residues on the photosystem I subunits PsaD and PsaE; von Leoprechting A et al.; An affinity purification technique was established that allows the selective isolation of 2-iminobiotinylated peptides from proteolytic digest of proteins in order to identify surface-exposed protein domains . Serving as model systems, two photosystem I subunits, PsaD and PsaE from the cyanobacterium Synechococcus elongatus, were overexpressed in Escherichia coli, modified in vitrowith NHS-2-iminobiotin which incorporates 2-iminobiotin at exposed amino groups, and subjected to proteolytic digestion by Glu-C and Arg-C protease, respectively . 2-Iminobiotin-containing proteolytic peptides were subsequently extracted from the proteolytic digests using avidin agarose in a batch procedure and the extracted peptides were separated by HPLC chromatography . The analysis of the peptide maps by electrospray ionization mass spectrometry or N-terminal sequencing showed that avidin-extracted peptide fractions contain almost exclusively 2-iminobiotinylated proteolytic fragments of PsaE or PsaD . No unmodified peptides of PsaD or PsaE were detected . According to this analysis, PsaE is accessible to biotinylation at all of its 7 lysine residues and at its N-terminus . Similarly, all 11 lysine residues of PsaD can be biotinylated and only the N-terminus of PsaD is not accessible . Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7441 - 5 Photorepair mutants of Arabidopsis; Jiang CZ et al.; UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product) . Although Escherichia coli and Saccharomyes cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosphila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products . We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively . We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase . In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers . This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system. Protein Eng, 1998 Aug, 11(8), 707 - 13 Effects of the length of a glycine linker connecting the N-and C-termini of a circularly permuted dihydrofolate reductase; Iwakura M et al.; An important consideration in the construction of active and stable circularly permuted proteins is the connective sequence that links the native N- and C-termini . For this reason, various lengths of polyglycine linkers (two, three, four, five and six glycines) were employed to connect the original N- and C-termini of a circularly permuted construct of Escherichia coli dihydrofolate reductase (DHFR) in which the new N-terminus was Met16 . Examination of the circular dichroism (CD) spectra, gel-filtration chromatography elution profiles, urea-induced unfolding properties and enzyme kinetics revealed that, among the linkers tested, a linker length of five glycines was the most favorable . The Vmax of the circularly permuted variant with a five glycine linker (cpM16G5) was about 20% that of wild-type DHFR, although far UV CD spectra, gel filtration elution time, conformational stability and Km for the substrate dihydrofolate and Kd for the coenzyme NADPH were comparable in the two proteins . Another circularly permuted DHFR with a five glycine linker in which a new N-terminus was created at Leu24 (cpL24G5) was also constructed and assayed . The Vmax of cpL24G5 was almost the same as the wild-type, presumably due to the optimization of the glycine linker . The improved activity of the Leu24 permutant is probably due to the disruption of a catalytically important structure, the M20 loop, in the Met16 permutant. Protein Eng, 1998 Aug, 11(8), 683 - 90 Calorimetric study of mutant human lysozymes with partially introduced Ca2+ binding sites and its efficient refolding system from inclusion bodies; Koshiba T et al.; During the process of evolution, ancestral lysozymes evolved into calcium-binding lysozymes by acquiring three critical aspartate residues at positions 86, 91 and 92 . To investigate the process of the acquisition of calcium-binding ability, two of the aspartates were partially introduced into human lysozyme at positions 86, 91 and 92 . These mutants (HLQ86D, HLA92D and HLQ86D/D91Q/A92D), having two critical aspartates in calcium-binding sites, were expressed in Escherichia coli as non-active inclusion bodies . For the preparation of lysozyme samples, a refolding system using thioredoxin was established . This system allowed for effective refolding of wild-type and mutant lysozymes, and 100% of activity was recovered within 4 days . The calcium ion dependence of the melting temperature (Tm) of wild-type and mutant lysozymes was investigated by differential scanning calorimetry at pH 4.5 . The Tm values of wild-type, HLQ86D and HLA92D mutants were not dependent on calcium ion concentration . However, the Tm of HLQ86D/D91Q/A92D was 4 degrees higher in the presence of 50 mM CaCl2 than in its absence, and the calcium-binding constant of this mutant was estimated to be 2.25(+/-0.25)x10(2) M(-1) at pH 4.5 . Moreover, the calcium-binding ability of this mutant was confirmed by the result using Sephadex G-25 gel chromatography . These results indicate that it is indispensable to have at least two aspartates at positions 86 and 92 for acquisition of calcium-binding ability . The process of the acquisition of calcium-binding site during evolution of calcium-binding lysozyme is discussed. Protein Eng, 1998 Aug, 11(8), 627 - 30 Misleading local sequence alignments: implications for comparative protein modelling; Saqi MA et al.; Although it is well known that significant sequence similarity between proteins is reflected at the structural level, it is commonly assumed that any misaligned regions, as judged by the correct structure based alignment, are those where the local sequence identity is lower than the global . Recent studies have shown that this is not always the case and there can exist short stretches of high local identity which is not reflected in the structure based alignment . An analysis is presented of 290 pairs of homologous proteins with a view to quantifying the occurrence of these misleading local sequence alignments (MLSAs) . It is found that such MLSAs are likely if the global sequence identity is less than 40% and can occur even when it is greater than 60% . The results have implications for automated homology modelling and also for the inference of function made by comparison. Mol Gen Genet, 1998 Aug, 259(3), 336 - 44 Orientation-dependent enhancement by H-NS of the activity of the type 1 fimbrial phase switch promoter in Escherichia coli; Schembri MA et al.; Phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a 314-bp DNA element, positioned proximal to and upstream of fimA, which encodes the major type 1 fimbrial subunit . This DNA switch region contains a promoter that drives transcription of fimA only when the switch is in the ON orientation . Using chromosomal and plasmid-borne lacZ reporter cassettes, we show here how the global regulator H-NS affects the activity of the fimA promoter . In phase-locked reporter cassettes the activity of the fimA promoter was found to be enhanced in a hns-positive background, but only when the switch was in the ON orientation . Also, the number of fimbriae produced by a phase-locked ON strain was significantly higher in a hns-positive background . By means of competitive gel retardation and DNase I protection assays, H-NS binding to DNA segments adjacent to and within the phase switch region was demonstrated. Mol Gen Genet, 1998 Aug, 259(3), 317 - 26 CRP down-regulates adenylate cyclase activity by reducing the level of phosphorylated IIA(Glc), the glucose-specific phosphotransferase protein, in Escherichia coli; Takahashi H et al.; The cellular cAMP level is markedly down-regulated by cAMP receptor protein (CRP) in Escherichia coli . CRP regulates adenylate cyclase both at the level of transcription of its structural gene cya and at the level of enzyme activity . We established a method to determine the phosphorylation state of IIA(Glc), the glucose-specific phosphotransferase protein, in intact cells . We found that IIA(Glc) exists predominantly in the unphosphorylated form in wild-type cells growing in LB medium, while it is largely phosphorylated in crp or cya cells . Disruption of the ptsG gene that codes for the membrane component of the major glucose transporter (IICB(Glc)), and/or the fruF gene coding for FPr (fructose-specific hybrid phosphotransferase protein), did not affect the phosphorylation state of IIA(Glc) . When IICB(Glc) was overproduced in the presence of glucose, the levels of both cAMP and phosphorylated IIA(Glc) in crp cells were concomitantly decreased to wild-type levels . In addition, when His-90 in IIA(Glc) was replaced by glutamine, both phosphorylation of IIA(Glc) and the overproduction of cAMP in crp cells were eliminated . We also found that extracts of crp+ cells markedly stimulate dephosphorylation of IIA(Glc)-P in vitro . We conclude that CRP-cAMP down-regulates adenylate cyclase primarily by reducing the level of phosphorylated IIA(Glc) . The data suggest that unspecified proteins whose expression is under the control of CRP-cAMP are responsible for this regulation. Am J Trop Med Hyg, 1998 Sep, 59(3), 347 - 51 Development of monoclonal antibodies specifically recognizing the cyst stage of Entamoeba histolytica; Walderich B et al.; Protozoan cysts were isolated according to a two-step sucrose gradient procedure . Pure cysts of Entamoeba histolytica, in fixed and native states, were injected into BALB/c mice intraperitoneally for immunization . The spleens of these animals were used for fusion with AG8 mouse myeloma cells . Hybridomas were obtained and tested for the recognition of E . histolytica, E . dispar, E . coli, E . hartmanni, Endolimax nana, Jodamoeba biitschlii, and Giardia lamblia . Three monoclonal antibodies were identified that reacted only with cysts and trophozoites of E . histolytica . These can be used for differentiation and identification of E . histolytica in feces. Mol Cells, 1998 Aug 31, 8(4), 483 - 90 Transcriptional control of the glnD gene is not dependent on nitrogen availability in Escherichia coli; Kim IH et al.; Glutamine synthetase (GS) is one of the most important enzymes in the assimilation of nitrogenous compounds in Escherichia coli and related bacteria . For the control of its activity and biosynthesis, tricyclic cascades of uridylylation/deuridylylation of PII protein, adenylylation/deadenylylation of glutamine synthetase, and phosphorylation/dephosphorylation of Ntr1 are operating, where the regulation of uridylylation/deuridylylation by uridylyl transferase-uridylyl removing enzyme (UT-UR) (the product of the glnD gene) would play the ultimate nitrogen sensing role . However, the possible nitrogen-regulatable element in the upstream of the glnD gene has been debated . In the present experiment, we have cloned and sequenced the four minute regions of the Escherichia coli chromosome, where rpsB, map, glnD, and dapD genes have been identified in sequence . We could localize the transcriptional start site at seven nucleotides upstream of the translation initiation codon by primer extension analysis . The nitrogen dependency of the glnD gene has been analyzed by Northern blot, RNase protection, and promoter-luciferase activity assays . These data suggested a constitutive expression of the glnD gene independent of nitrogen availability . From these results, it could be concluded that the ultimate nitrogen sensing device for the bacteria should be the UT-UR itself, through modulation of its activity in response to the nitrogen status rather than its biosynthetic mechanism. Mol Cells, 1998 Aug 31, 8(4), 478 - 82 Expression, purification, and characterization of a familial amyotrophic lateral sclerosis-associated D90A Cu,Zn-superoxide dismutase mutant; Kim SM et al.; Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS) . We cloned the FALS mutant, D90A, and wild-type of human Cu,Zn-SOD, overexpressed them in E . coli, purified the proteins, and studied their properties . We investigated their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as a substrate . Our results showed that both wild-type and mutant enzymes have identical dismutation activities . However, the hydroxyl radical-generating function of the D90A mutant, as measured using a 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate), was enhanced relative to that of the wild-type enzyme . Catalysis of this reaction by D90A was more sensitive to inhibition by the copper chelators, penicillamine and diethyldithiocarbamate, than was catalysis by wild-type Cu,Zn-SOD . Our study suggests that this gain-of-function of FALS mutant may, in part, be responsible for the development of FALS symptoms. Mol Cells, 1998 Aug 31, 8(4), 438 - 43 Cloning, nucleotide sequence, and expression of the DNA ligase-encoding gene from Thermus filiformis; Kim HK et al.; The gene encoding Thermus filiformis (Tfi) DNA ligase was cloned and its nucleotide sequence was determined by the chain-termination method . The primary structure of Tfi DNA ligase was deduced from its nucleotide sequence . The Tfi DNA ligase comprises of 667 amino acid residues and its molecular mass was determined to be 75,936 Da . The deduced amino acid sequence of Tfi DNA ligase showed a 86.5% homology to Tth DNA ligase and 43.5% to E . coli DNA ligase . The Lys-116 of Lys-Val-Asp-Gly motif was proposed to be the active residue of Tfi DNA ligase . In comparison with the amino acid composition of DNA ligase, Tfi DNA ligase showed a significant increase in the proportion of charged residues, Arg and Glu, compared to E . coli DNA ligase . The G + C content in the first, second, and third positions of the codons used were 70.3%, 40.3%, and 90.3%, respectively . Codon usage in Tfi DNA ligase was heavily biased towards the use of G + C in the third position . Under tac promoter control, Tfi DNA ligase was overproduced to greater than 9% of E . coli BL26Blue cellular proteins. Mol Cells, 1998 Aug 31, 8(4), 416 - 23 Overexpression and characterization of the cDNA encoding the coat protein of cucumber mosaic virus (Strain ABI) isolated in Korea; Kim YM et al.; We constructed a recombinant CMVCP expression vector termed pMALCMV in which cDNA fragment encoding CMVCP is ligated into pMAL-c2, an E . coli expression vector . Overexpression of pMALCMV containing the entire open reading frame of CMV cDNA sequence and the maltose binding protein (MBP) leader gene was facilitated in E . coli TB1 cells, which resulted in the production of a fusion protein of MBP-CMVCP (Mr 67.7 kDa) that was immunoprecipitable with rabbit polyclonal antiserum specific for MBP . The CMVCP (Mr 24.5 kDa) was isolated through a preparative SDS polyacrylamide gel following digestion of the affinity ligand purified fusion protein with Factor Xa . The partial amino acid sequences of the cleaved proteins were confirmed at the amino terminus by peptide sequencing . The CMVCP antiserum was also prepared by intraperitoneal injection of this purified CP into a BALB/c mouse . Immunoblot analysis showed that the purified CMVCP from the Factor Xa cleavage reaction was an authentic overexpression product of the cloned CMVCP . Using an RNA mobility shift assay, it was demonstrated that CMVCP can bind to its own RNA transcript in a concentration dependent manner . However, the complex formed between CMVCP and its RNA was abolished by the addition of a polyclonal antibody that had been raised against CMVCP, confirming that the overexpressed CMVCP specifically interacts with its own RNA . Thus, our results can provide a basis for the development of a hybridoma cell line expressing the monoclonal antibody for CMVCP and molecular cloning of their genes, which may lead to the creation of CMV-resistant transgenic plants. J Dairy Sci, 1998 Aug, 81(8), 2159 - 64 Influence of route of vaccine administration against experimental intramammary infection caused by Escherichia coli; Tomita GM et al.; The route of immunization of a commercially available Escherichia coli J5 bacterin was investigated . Jersey cows were randomly assigned to one of three treatment groups: 1) unvaccinated (control), 2) vaccinated subcutaneously in the neck, and 3) vaccinated in the area of the supramammary lymph node . Cows were vaccinated at drying off and at 2 wk prior to anticipated calving . Two quarters of each cow were challenged with approximately 60 cfu of E . coli at 14 d postcalving . Route of immunization in the neck or the area of the supramammary lymph node did not influence severity of coliform mastitis . However, the mean number of colony-forming units of E . coli recovered from challenged quarters was significantly lower for vaccinated cows than for control cows at 24 h postchallenge . A quicker milk yield recovery following intramammary challenge was also observed for vaccinated cows . Serum immunoglobulin (Ig) G, IgG1, and IgG2 and whey IgG1 and IgG2 antibody titers against E . coli J5 whole-cell antigens were significantly enhanced in vaccinated cows . Somatic cell counts in milk from challenged quarters and rectal temperatures following intramammary challenge were not different for cows across treatment groups . Immunization did not prevent intramammary infection. Genetika, 1998 Jul, 34(7), 1017 - 20 {Eukaryotic DNA topoisomerase and in vitro recombination between circular supercoiled plasmid DNA's in an in vitro system}; Glazkov MV et al.; Eukaryotic DNA topoisomerase II was shown to catalyze recombination between circular supercoiled plasmid DNAs in vitro . The results obtained are discussed with regard to the organization of eukaryotic chromosomes in the form of topologically independent domains (loops). Bioorg Khim, 1998 Jul, 24(7), 530 - 8 {Regulation of synthesis of ribosomal protein L7-L12: role of intercistronic rplJL region as an enhancer of translation}; Artamonova VS et al.; The ability of the Escherichia coli intercistronic rplJL region to initiate effectively the synthesis of the ribosomal protein L7/12, the only ribosomal component present in the ribosome in four copies rather than in one was studied in vivo and in vitro . It was shown that the structural determinants located upstream from the Shine-Dalgarno sequence and sharing structural motifs with the known E . coli translational enhancers are necessary for high activity of this region in translation initiation . These data indicate that mRNA-protein interactions through the ribosomal S1 protein play an important role in the formation of the initiation complex, and an enhancer region within the leader of the L7/12 mRNA serves as a target for this protein. Bioorg Khim, 1998 Jul, 24(7), 523 - 9 {Production of recombinant human keratinocyte growth factor in Escherichia coli cells}; Ptitsyn LR et al.; Here we describe the synthesis of the gene encoding human keratinocyte growth factor (hKGF) and the construction of vectors for cytoplasmic production of hKGF in Escherichia coli cells . The level of recombinant protein expression was 1-1.5% of the total cell protein . hKGF was purified to homogeneity by three-step ion-exchange and affinity chromatography . The protein is biologically active: it stimulates dose-dependent protein synthesis in cultured human epidermal keratinocytes. Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 462 - 8 Carbamoyl-phosphate synthetase II in kinetoplastids; Nara T et al.; Genes for carbamoyl-phosphate synthetase II (CPS II), the first enzyme of de novo pyrimidine biosynthesis, were cloned from kinetoplastids, Trypanosoma cruzi and Leishmania mexicana . T . cruzi CPS II gene encodes a protein of 1524 amino acids that encompasses the glutaminase and CPS domains, but incorporates neither aspartate carbamoyltransferase nor dihydroorotase . The residue corresponding to lysine 993 of Escherichia coli CPS, a residue that characterizes the CPS inhibited by UMP and that is replaced by tryptophan in those inhibited by UTP, is in kinetoplastids a hydrophilic glutamine, in line with the preferential inhibition by UDP of kinetoplastid CPS II. Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 422 - 32 Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein; Wu H et al.; A 429 aa theoretical intein is encoded in the dnaB gene (DNA helicase) of the cyanobacterium Synechocystis sp . strain PCC6803 . This intein is shown to be capable of protein splicing with or without its native exteins when tested in E . coli cells . A centrally located 275 amino acid sequence (residues 107-381) of this intein can be deleted without loss of the protein splicing activity, resulting in a functional mini-intein of 154 aa in size . Efficient in vivo protein trans-splicing was observed when this mini-intein was split into a 106 aa N-terminal fragment containing intein motifs A and B, and a 48 aa C-terminal fragment containing intein motifs F and G . These results indicate that the N- and C-terminal regions of the Ssp DnaB intein, whether covalently linked with each other or not, can come together through non-covalent interaction to form a protein splicing domain that is functionally sufficient and structurally independent from the centrally located endonuclease domain of the intein. Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 387 - 94 Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics; Peake P et al.; We have examined the functional properties including autophosphorylation of the Mycobacterium leprae Hsp70 homologue . Recombinant M . leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphorylating activities which were near pH 8 and 6, respectively . Both these activities were inhibited by reduced and alkylated bovine pancreatic trypsin inhibitor, but not other tested substrates . Autophosphorylation was augmented by up to 25 mM Ca2+ . Using site-directed mutagenesis to construct two Thr-->Ala mutants at positions 175 and 193, and phosphoamino acid analysis, it was shown that Thr175 was the dominant threonine residue autophosphorylated in M . leprae Hsp70 . Phosphorylation led to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin . These properties are compared with those of the DnaK protein of Escherichia coli. Biochim Biophys Acta, 1998 Aug 28, 1393(2-3), 267 - 76 A phospholipase A2 is transiently synthesized during seed germination and localized to lipid bodies; May C et al.; A patatin-like protein is present in the storage tissue of cucumber seedlings during the stage of fat mobilization . The cucumber protein is a homologue of a glycoprotein which in potatoes accounts for most of the total protein content of tubers . Following preparation of a cucumber cDNA library representing the developmental stage of cotyledons of 1 day old germinating seeds we isolated and characterized a clone encoding a patatin-like protein . Antibodies raised against the protein expressed in bacteria were used for immunodetection in subcellular fractions of cucumber seedlings . It was shown that the patatin-like protein was virtually exclusively confined to lipid bodies . The protein expressed in bacteria was characterized in vitro by its esterase activity acting on monoacylglycerols and phospholipids . Detailed analysis using various forms of phosphatidyl choline as substrates demonstrated that the patatin-like protein is a phospholipase A2 acting on palmitoyl, linoleoyl and hydroperoxidized linoleoyl groups equally well . Studying the temporal and tissue-specific expression of patatin-like protein mRNA we showed its appearance exclusively during fat catabolism . As maximal amounts of the protein were found at an early stage of mobilization and confined to lipid bodies, we propose that the patatin-like hydrolase is involved in lipid body mobilization. Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 136 - 42 The role of tryptophan residues in Escherichia coli arginyl-tRNA synthetase; Zhang QS et al.; The effect of N-bromosuccinimide (NBS) on the activity of Escherichia coli arginyl-tRNA synthetase (ArgRS) was studied . The results showed that only one tryptophan residue was easy of access to the reagent and was closely related to enzyme activity . When all the five tryptophan residues in ArgRS were changed via site-directed mutagenesis singly into Ala, the aminoacylation activity of the Trp162 mutated enzyme decreased seriously, while the other four mutant enzymes retained almost the same activity as the native one . The oxidation of the five mutant enzymes with NBS demonstrated that only the mutation of Trp162 resulted in the loss of sensitivity to the reagent . These results strongly suggest that Trp162 is more accessible to NBS and is related to enzyme activity . Furthermore, the far-UV CD spectroscopy of the mutant enzyme ArgRS162WA showed little change in its secondary structure . Finally, studies on the kinetics of the mutant enzyme ArgRS162WA in aminoacylation reaction showed that the reduction in activity could be attributed to the decrease in the values of kcat and kcat/Km for arginine . The thermodynamic calculation indicates that this mutation causes a decrease of the binding energy by 2.7 kJ/mol . Our data suggest that Trp162 is involved in the binding of arginine and in the transition state stabilization. Mutat Res, 1998 Oct 12, 421(1), 121 - 36 A comparative study of in vivo mutation assays: analysis of hprt, lacI, cII/cI and as mutational targets for N-nitroso-N-methylurea and benzo{a}pyrene in Big Blue mice; Monroe JJ et al.; We have compared the response of the native hprt gene and the lacI, cII, and cI transgenes in Big Blue B6C3F1 mice following treatment with either N-nitroso-N-methylurea (MNU) or benzo{a}pyrene (BaP) . Three weeks after mutagen treatment splenic T cells were isolated from the animals, and samples were either cultured to measure mutation at the native hprt locus or used to extract genomic DNA for transgene mutation analysis . Phage rescued from extracted DNA were plated in the presence of 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) to score lacI mutations, or plated on a hflAB lawn to score cII and cI mutants . With MNU hprt mutant frequency increased in a dose-related, sublinear manner up to 78-fold above background at the highest dose tested (20 mg/kg) . In comparison, the lacI transgene yielded only a 3.1-fold increase at this dose, and the cII and cI transgenes did not show any increase . With 150 mg/kg BaP a 5.8- and 8.7-fold increase in mutant frequency was observed at hprt and lacI, respectively, while only a 1.3-fold increase was observed at cII . DNA sequencing revealed an increase in GC-->TA transversions among the cII mutants, suggesting that the increase was related to BaP exposure . No significant increase in cI mutant frequency was observed . Therefore, the order of mutation assay sensitivity was hprt>lacI>cII/cI with MNU, and hprt approximately lacI> cII/cI with BaP . While the hflAB selection system offers significant advantages with respect to cost and effort when compared to the lacI assay, additional evaluation of its sensitivity is warranted . Biochim Biophys Acta, 1998 Oct 1, 1400(1-3), 29 - 43 DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities; Levine C et al.; DNA gyrase and topoisomerase IV are the two type II topoisomerases present in bacteria . Though clearly related, based on amino acid sequence similarity, they each play crucial, but distinct, roles in the cell . Gyrase is involved primarily in supporting nascent chain elongation during replication of the chromosome, whereas topoisomerase IV separates the topologically linked daughter chromosomes during the terminal stage of DNA replication . These different roles can be attributed to differences in the biochemical properties of the two enzymes . The biochemical activities, physiological roles, and drug sensitivities of the enzymes are reviewed. Biochim Biophys Acta, 1998 Oct 1, 1400(1-3), 3 - 18 Structure of DNA topoisomerases; Berger JM; Over the last several years topoisomerases have finally begun to yield to high-resolution structural studies . These models have greatly aided our understanding of the mechanisms of topoisomerase catalysis and drug interactions . This review will cover advances in the structural biology of topoisomerases and discuss their implications for topoisomerase function. J Bacteriol, 1998 Oct, 180(19), 5279 - 83 Two amino acid residues of transposase contributing to differential transposability of IS1 elements in Escherichia coli; Chen JH et al.; Escherichia coli W3110 contains four types of IS1 elements in the chromosome . Using an insertion element entrapping system, we collected 116 IS1 plasmid insertion mutants, which resulted from a minimum of 26 independent IS1 insertion events . All of them had insertions of IS1 of the IS1A (IS1E and IS1G) type . Inspection of the transposase sequences of the four IS1 types and the IS1 of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS1 elements in W3110 . The two amino acid residues of the transposase in IS1A (IS1E and IS1G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS1A (IS1E and IS1G) . Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS1 elements in W3110 was confirmed. J Bacteriol, 1998 Oct, 180(19), 5243 - 6 The gene for 16S rRNA methyltransferase (ksgA) functions as a multicopy suppressor for a cold-sensitive mutant of era, an essential RAS-like GTP-binding protein in Escherichia coli; Lu Q et al.; Era, a Ras-like GTP-binding protein in Escherichia coli, has been shown to be essential for growth . However, its cellular functions still remain elusive . In this study, a genetic screening of an E . coli genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid . Among eight suppressors isolated, six were located at 1 min of the E . coli genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the ksgA gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor . This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme . A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed. J Bacteriol, 1998 Oct, 180(19), 5240 - 2 Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an intact gene X signal sequence and an active translocon; Oliver D et al.; secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell . This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling . Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation . These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation . Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration . We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor. J Bacteriol, 1998 Oct, 180(19), 5173 - 82 ClpB in a cyanobacterium: predicted structure, phylogenetic relationships, and regulation by light and temperature; Celerin M et al.; The sequence of a genomic clone encoding a 100-kDa stress protein of Plectonema boryanum (p-ClpB) was determined . The predicted polypeptide contains two putative ATPase regions located within two highly conserved domains (N1 and N2), a spacer region that likely forms a coiled-coil domain, and a highly conserved consensus CK2 phosphorylation domain . The coiled-coil region and the putative site of phosphorylation are not unique to p-ClpB; they are present in all ClpB sequences examined and are absent from the ClpB paralogs ClpA, ClpC, ClpX, and ClpY . Small quantities of a 4.5-kb p-clpB transcript and 110-kDa cytosolic p-ClpB protein were detected in cells grown under optimal conditions; however, increases in the quantities of the transcript and protein were observed in cells grown under excess light and low temperature conditions . Finally, we analyzed ClpA, ClpB, and ClpC sequences from 27 organisms in order to predict phylogenetic relationships among the homologs . We have used this information, along with an identity alignment, to redefine the Clp subfamilies. J Bacteriol, 1998 Oct, 180(19), 5165 - 72 Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA, ClpB, and HtpG In vivo; Thomas JG et al.; We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene . All mutants exhibited growth defects at 46 degrees C, but not at lower temperatures . However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50 degrees C . When cultures were allowed to recover from transient exposure to 50 degrees C, all mutations except Deltaibp led to suboptimal growth as the recovery temperature was raised . Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42 degrees C when combined with the dnaK756 or groES30 alleles, while the Deltaibp mutation had a detrimental effect only on the growth of dnaK756 mutants . Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures . Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46 degreesC compared to wild-type cells, none of the null mutations had a similar effect . These results show that the highly conserved E . coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures . Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E . coli chaperone systems in vivo. J Bacteriol, 1998 Oct, 180(19), 5123 - 8 CheZ has no effect on flagellar motors activated by CheY13DK106YW; Scharf BE et al.; The behaviors of both cheZ-deleted and wild-type cells of Escherichia coli were found to be very sensitive to the level of expression of CheZ, a protein known to accelerate the dephosphorylation of the response regulator CheY-phosphate (CheY-P) . However, cells induced to run and tumble by the unphosphorylated mutant protein CheY13DK106YW (CheY**) failed to respond to CheZ, even when CheZ was expressed at high levels . Therefore, CheZ neither affects the flagellar motors directly nor sequesters CheY** . In in vitro cross-linking studies, CheY** promoted trimerization of CheZ to the same extent as wild-type CheY but failed to induce the formation of complexes of higher molecular weight observed with CheY-P . Also, CheY** could be cross-linked to FliM, the motor receptor protein, nearly as well as CheY-P . Thus, to CheZ, CheY** looks like CheY, but to FliM, it looks like CheY-P. J Bacteriol, 1998 Oct, 180(19), 5109 - 16 Effect of slow growth on metabolism of Escherichia coli, as revealed by global metabolite pool ("metabolome") analysis; Tweeddale H et al.; Escherichia coli growing on glucose in minimal medium controls its metabolite pools in response to environmental conditions . The extent of pool changes was followed through two-dimensional thin-layer chromatography of all 14C-glucose labelled compounds extracted from bacteria . The patterns of metabolites and spot intensities detected by phosphorimaging were found to reproducibly differ depending on culture conditions . Clear trends were apparent in the pool sizes of several of the 70 most abundant metabolites extracted from bacteria growing in glucose-limited chemostats at different growth rates . The pools of glutamate, aspartate, trehalose, and adenosine as well as UDP-sugars and putrescine changed markedly . The data on pools observed by two-dimensional thin-layer chromatography were confirmed for amino acids by independent analysis . Other unidentified metabolites also displayed different spot intensities under various conditions, with four trend patterns depending on growth rate . As RpoS controls a number of metabolic genes in response to nutrient limitation, an rpoS mutant was also analyzed for metabolite pools . The mutant had altered metabolite profiles, but only some of the changes at slow growth rates were ascribable to the known control of metabolic genes by RpoS . These results indicate that total metabolite pool ("metabolome") analysis offers a means of revealing novel aspects of cellular metabolism and global regulation. J Bacteriol, 1998 Oct, 180(19), 5102 - 8 Expression of the Kdp ATPase is consistent with regulation by turgor pressure; Malli R et al.; The kdpFABC operon of Escherichia coli encodes the four protein subunits of the Kdp K+ transport system . Kdp is expressed when growth is limited by the availability of K+ . Expression of Kdp is dependent on the products of the adjacent kdpDE operon, which encodes a pair of two-component regulators . Studies with kdp-lac fusions led to the suggestion that change in turgor pressure acts as the signal to express Kdp (L . A . Laimins, D . B . Rhoads, and W . Epstein, Proc . Natl . Acad . Sci . USA 78:464-468, 1981) . More recently, effects of compatible solutes, among others, have been interpreted as inconsistent with the turgor model (H . Asha and J . Gowrishankar, J . Bacteriol . 175:4528-4537, 1993) . We re-examined the effects of compatible solutes and of medium pH on expression of Kdp in studies in which growth rate was also measured . In all cases, Kdp expression correlated with the K+ concentration when growth began to slow . Making the reasonable but currently untestable assumptions that the reduction in growth rate by K+ limitation is due to a reduction in turgor and that addition of betaine does not increase turgor, we concluded that all of the data on Kdp expression are consistent with control by turgor pressure. Biochemistry, 1998 Sep 22, 37(38), 13269 - 75 Ligand-induced conformational transitions in Escherichia coli phosphofructokinase 2: evidence for an allosteric site for MgATP2-; Guixe V et al.; The binding of ligands to phosphofructokinase 2 (Pfk-2) from Escherichia coli induces changes in the fluorescence emission properties of its single tryptophan residue, Trp88, suggesting that upon binding the protein undergoes a conformational change . This fluorescence probe was used to determine the presence of an allosteric site for MgATP2- in the enzyme . Fructose 6-phosphate (fructose-6-P), the first substrate that binds to the enzyme with an ordered bi-bi mechanism, increases the fluorescence up to 30% . The saturation curve for this compound is hyperbolic with a Kd of 6 microM . The titration of Pfk-2 with MgATP2- causes a quenching of fluorescence of about 30% of its initial value, with a blue shift of 7 nm in the emission maximum . The response is cooperative with a Kd of 80 microM and a Hill coefficient of 2 . The interaction of MgATP2- cannot take place at the active site in the absence of fructose-6-P, due to the ordered kinetic mechanism . Addition of compounds that bind to the catalytic site of Pfk-2, such as ATP4- or Mg-AMP-PNP, did not produce significant changes in the fluorescence spectrum of Trp88 . However, in the absence of Mg2+, the addition of ATP4- to the enzyme-fructose-6-P complex shows a hyperbolic increase of fluorescence of 8% . Acrylamide steady-state quenching experiments for different enzyme-ligand complexes of Pfk-2, indicate that the tryptophan in the enzyme-MgATP2- complex is exposed to a much smaller extent to the solvent than in the free enzyme or in the enzyme-fructose-6-P complex . The effect of the binding of fructose-6-P or MgATP2- on the polarization fluorescence of the emission of Trp88 in Pfk-2 indicates changes in the local mobility of the Trp88 in both enzyme complexes . The average lifetime for Trp88 in Pfk-2 was found to be unusually large, approximately 7.7 ns, and did not vary significantly with the ligation state of the enzyme, which demonstrates that the quenching or enhancement of fluorescence induced by the ligands is mainly due to the complex formation with Pfk-2 . These results demonstrate the presence of an allosteric site for MgATP2- in Pfk-2 from E . coli, responsible for the inhibition of the enzyme activity by this ligand. Biochemistry, 1998 Sep 22, 37(38), 13230 - 8 Kinetic mechanism of Escherichia coli asparagine synthetase B; Boehlein SK et al.; Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP . A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B . The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia . The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding . However, product inhibition studies were only consistent with the latter pathway . Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent . Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source . Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding . This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer. Biochemistry, 1998 Sep 22, 37(38), 13212 - 21 Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine; Curien G et al.; Plant threonine synthase, in contrast to its bacterial counterpart, is strongly stimulated by S-adenosylmethionine via a noncovalent interaction {Giovanelli et al . (1984) Plant . Physiol . 76, 285-292} . The mechanism of activation remained, however, largely unknown . To further characterize this unusual role for S-adenosylmethionine, the Arabidopsis thaliana threonine synthase was overexpressed in Escherichia coli, purified to homogeneity, and then used for kinetic and enzyme-bound pyridoxal 5'-phosphate fluorescence equilibrium-binding experiments . We observed that the activating effect of S-adenosylmethionine results from an 8-fold increase in the rate of catalysis and from a 25-fold decrease in the Km value for the O-phosphohomoserine substrate . The data can be well fitted by a kinetic model assuming binding of two S-adenosylmethionine molecules on the native enzyme . We suggest that the dramatic modifications of the enzyme kinetic properties originate most presumably from an allosteric and cooperative transition induced by S-adenosylmethionine . This transition occurs at a much faster rate in the presence of the substrate than in its absence. World J Surg, 1998 Oct, 22(10), 1069 - 75; discussion 1076 Wound healing of intestinal anastomosis after digestive surgery under septic conditions: participation of local interleukin-6 expression; Ishimura K et al.; This study aimed to evaluate the integrity of anastomotic wound healing after digestive surgery under septic conditions and to observe local interleukin-6 (IL-6) expression around the anastomotic segment . Experimental animals were separated into lipopolysaccharide (LPS) and control groups . Each was injected with LPS or normal saline solution into the peritoneal cavity 24 hours before transection and anastomosis of the colon . The anastomotic bursting pressure (ABP) and tissue hydroxyproline concentration (HP) were measured as indicators of wound healing . Immunohistochemical staining for IL-6 was performed on tissue samples obtained from the anastomotic segment, lung, liver, and kidney . The reactive cells were counted by light microscopy . The ABP and HP were significantly lower in the LPS group than the control group 7 days after the surgery . In the LPS group, IL-6 expression around the anastomotic segment was enhanced 1 and 6 hours after surgery but suppressed 24 hours afterward . In contrast, IL-6 expression in lung, liver, and kidney was enhanced in the LPS group 24 hours after surgery but not in the control group . It is suggested that anastomotic wound healing is impaired after digestive tract surgery under septic conditions, and local IL-6 expression participates in wound healing. J Biol Chem, 1998 Oct 2, 273(40), 26130 - 7 Cloning and characterization of the 5'-flanking region for the human topoisomerase III gene; Kim JC et al.; The human DNA topoisomerase III (hTOP3) gene encodes a topoisomerase homologous to the Escherichia coli DNA topoisomerase I subfamily . To understand the mechanisms responsible for regulating hTOP3 expression, we have cloned the 5'-flanking region of the gene coding for the hTOP3 and analyzed its promoter activity . The presence of a single transcription initiation site was suggested by primer extension analysis . The hTOP3 gene promoter is moderately high in GC content and lacks a canonical TATA box, suggesting that hTOP3 promoter has overall similarity to promoters of a number of housekeeping genes . Examination of the promoter sequence indicated the presence of four Sp-1 consensus binding sequences and a putative initiator element surrounding the transcription initiation site . Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 52-base pair region from -326 to -275 upstream of the transcription initiation site includes a positive cis-acting element(s) for the efficient expression of hTOP3 gene . On the basis of gel mobility shift and supershift assays, we demonstrated that both YY1 and USF1 transcription factors can bind to the 52-base pair region . When HeLa cells were transiently transfected with a mutant construct which had disabled both YY1- and USF1-binding sites, the luciferase activity was greatly reduced, suggesting that these binding elements play a functional role in the basal activation of the hTOP3 promoter . Transfection studies with mutations that selectively impaired YY1 or USF1 binding suggested that both YY1 and USF1 function as activators in the hTOP3 promoter. J Biol Chem, 1998 Oct 2, 273(40), 25974 - 86 Catabolism of phenylacetic acid in Escherichia coli . Characterization of a new aerobic hybrid pathway; Ferrandez A et al.; The paa cluster of Escherichia coli W involved in the aerobic catabolism of phenylacetic acid (PA) has been cloned and sequenced . It was shown to map at min 31.0 of the chromosome at the right end of the mao region responsible for the transformation of 2-phenylethylamine into PA . The 14 paa genes are organized in three transcription units: paaZ and paaABCDEFGHIJK, encoding catabolic genes; and paaXY, containing the paaX regulatory gene . The paaK gene codes for a phenylacetyl-CoA ligase that catalyzes the activation of PA to phenylacetyl-CoA (PA-CoA) . The paaABCDE gene products, which may constitute a multicomponent oxygenase, are involved in PA-CoA hydroxylation . The PaaZ protein appears to catalyze the third enzymatic step, with the paaFGHIJ gene products, which show significant similarity to fatty acid beta-oxidation enzymes, likely involved in further mineralization to Krebs cycle intermediates . Three promoters, Pz, Pa, and Px, driven the expression of genes paaZ, paaABCDEFGHIJK, and paaX, respectively, have been identified . The Pa promoter is negatively controlled by the paaX gene product . As PA-CoA is the true inducer, PaaX becomes the first regulator of an aromatic catabolic pathway that responds to a CoA derivative . The aerobic catabolism of PA in E . coli represents a novel hybrid pathway that could be a widespread way of PA catabolism in bacteria. J Biol Chem, 1998 Oct 2, 273(40), 25893 - 902 Sequence characteristics, subcellular localization, and substrate specificity of DYRK-related kinases, a novel family of dual specificity protein kinases; Becker W et al.; DYRK1 is a dual specificity protein kinase presumably involved in brain development . Here we show that the kinase belongs to a new family of protein kinases comprising at least seven mammalian isoforms (DYRK1A, DYRK1B, DYRK1C, DYRK2, DYRK3, DYRK4A, and DYRK4B), the yeast homolog Yak1p, and the Drosophila kinase minibrain (MNB) . In rat tissues, DYRK1A is expressed ubiquitously, whereas transcripts for DYRK1B, DYRK2, DYRK3, and DYRK4 were detected predominantly in testes of adult but not prepuberal rats . By fluorescence microscopy and subcellular fractionation, a green fluorescent protein (GFP) fusion protein of DYRK1A was found to accumulate in the nucleus of transfected COS-7 and HEK293 cells, whereas GFP-DYRK2 was predominantly detected in the cytoplasm . DYRK1A exhibited a punctate pattern of GFP fluorescence inside the nucleus and was co-purified with the nuclear matrix . Analysis of GFP-DYRK1A deletion constructs showed that the nuclear localization of DYRK1A was mediated by its nuclear targeting signal (amino acids 105-139) but that its characteristic subnuclear distribution depended on additional N-terminal elements (amino acids 1-104) . When expressed in Escherichia coli, DYRK1A, DYRK2, DYRK3, MNB, and Yak1p catalyzed their autophosphorylation on tyrosine residues . The kinases differed in their substrate specificity in that DYRK2 and DYRK3, but not DYRK1A and MNB, catalyzed phosphorylation of histone H2B . The heterogeneity of their subcellular localization and substrate specificity suggests that the kinases are involved in different cellular functions. J Biol Chem, 1998 Oct 2, 273(40), 25809 - 17 Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases; Landis DM et al.; Thymidylate synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemotherapeutic agent 5-fluorouracil . We used random sequence mutagenesis to replace 13 codons within the active site of TS and obtain variants that are resistant to 5-fluorodeoxyuridine (5-FdUR) . The resulting random library was selected for its ability to complement a TS-deficient Escherichia coli strain, and sequence analysis of survivors found multiple substitutions to be tolerable within the targeted region . An independent selection of the library was carried out in the presence of 5-FdUR, resulting in a more limited spectrum of mutations . One specific mutation, C199L, was observed in more than 46% of 5-FdUR-resistant clones . A 5-FdUR-resistant triple mutant, A197V/L198I/C199F, was purified to apparent homogeneity . Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate . In contrast, equilibrium binding studies with the inhibitor, FdUMP, demonstrate that the dissociation constant (Kd) for FdUMP binding into the ternary complex was 20-fold higher than values obtained for the wild-type enzyme . This 5-FdUMP-resistant mutant, or others similarly selected, is a candidate for use in gene therapy to render susceptible normal cells resistant to the toxic effects of systemic 5-fluorouracil. J Biol Chem, 1998 Oct 2, 273(40), 25741 - 4 Superoxide dependence of the toxicity of short chain sugars; Benov L et al.; Erythrose inhibited the growth of a sodA sodB strain of Escherichia coli under aerobiosis; but did not inhibit anaerobic growth of the sodA sodB strain, or the aerobic growth of the superoxide dismutase (SOD)-competent parental strain . A SOD mimic protected the sodA sodB strain against the toxicity of erythrose as did the carbonyl-blocking reagents hydrazine and aminoguanidine . Three carbon sugars, such as glyceraldehyde and dihydroxy acetone, and the two carbon sugar glycolaldehyde, were similarly toxic in an O-2-dependent manner . An unidentified dialyzable component in E . coli extract augmented the oxidation of short chain sugars, and this was partially inhibitable by SOD . The toxicity of the short chain sugars appears to be because of an O-2-dependent oxidation to alpha, beta-dicarbonyl compounds . In keeping with this view was the O-2-independent toxicity of methylglyoxal. J Biol Chem, 1998 Oct 2, 273(40), 25594 - 601 Phenylalanine hydroxylase from Chromobacterium violaceum . Uncoupled oxidation of tetrahydropterin and the role of iron in hyroxylation; Chen D et al.; A gene encoding phenylalanine hydroxylase has been cloned from Chromobacterium violaceum and expressed in Escherichia coli . The purified phenylalanine hydroxylase contains copper, which does not support enzymatic activity . Upon removal of copper by dithiothreitol (DTT), the enzyme contains substoichiometric amounts of calcium and zinc but little or no redox-active metal ions . The copper-depleted hydroxylase catalyzes the phenylalanine-dependent oxidation of 6, 7-dimethyltetrahydropterin (DMPH4) by O2 in a reaction in which phenylalanine is not hydroxylated and does not appear to undergo a chemical change, and hydrogen peroxide is produced . Analogs of phenylalanine also activate the oxidation of DMPH4 . Both the copper-phenylalanine hydroxylase and the copper-depleted hydroxylase catalyze the hydroxylation of phenylalanine in the presence of DTT and FeSO4 in a reaction in which hydrogen peroxide is not produced . The apparent values of Km for Fe2+ and DTT are 0.28 microM and 1.1 mM, respectively, at 1.0 mM phenylalanine, 120 microM DMPH4 and pH 7 . 4 and 23 degreesC . The apparent value of kcat is 14.3 s-1 under these conditions . Glutathione, mercaptoethanol, and dihydrolipoate support the hydroxylation of phenylalanine essentially as well as DTT . Incubation of copper-depleted hydroxylase with FeSO4, phenylalanine, and DTT followed by gel permeation chromatography leads to an iron-hydroxylase containing approximately 1 molecule of iron per molecule of enzyme . The iron-hydroxylase displays an optical absorption band extending from 300 to 600 nm, and it catalyzes the hydroxylation of phenylalanine at the same maximum rate as the iron-activated hydroxylase but does not require added Fe2+ . We conclude that iron participates in the hydroxylation of phenylalanine . Iron is not required for the oxidation of DMPH4, although it may exert a modest acceleration effect . A hypothetical mechanism is presented wherein the reaction of iron with the putative 4a-hydroperoxy-DMPH4 leads to 4a-hydroxy-DMPH4 and a high valent iron-oxy species . The iron-oxy species is postulated to react with phenylalanine in the hydroxylation process. Plant Mol Biol, 1998 Oct, 38(3), 481 - 90 Tissue- and stage-specific expression of a soybean (Glycine max L.) seed-maturation, biotinylated protein; Hsing YC et al.; A cDNA clone GmPM4 which encodes mRNA species in mature or dry soybean seeds was characterized . DNA sequence analysis shows that the deduced polypeptides have a molecular mass of 68 kDa . GmPM4 proteins have a relatively high amino acid sequence homology with a major biotinylated protein isolated from pea seeds, SBP65, but both of these proteins differ markedly from that of presently known biotin enzymes . The accumulation of GmPM4 mRNA is detectable in the leaf primodium and the vascular tissues of the hypocotyl-radicle axis of mature seeds, and the GmPM4 proteins are present at high levels in dry and mature soybean seeds, but not in fresh immature seeds . It degrades rapidly at the early stage of seed germination . These proteins are boiling-soluble and biotinylated when they are present endogenously in soybean seeds; however, the same recombinant protein expressed in Escherichia coli is boiling-soluble, but it is not biotinylated. Plant Mol Biol, 1998 Oct, 38(3), 347 - 56 The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots; Tzafrir I et al.; Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity . The genomic region spanning nucleotides 5999-7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana . In A . sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive . In A . thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique . The transgene was inherited and active in progeny plants of both A . sativa and A . thaliana. Plant Mol Biol, 1998 Nov 1, 38(4), 513 - 20 Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development; Meng H et al.; Seasonal expression of caffeoyl-CoA O-methyltransferase (EC 2.1.1.104) was analyzed in aspen developing secondary xylem in parallel with caffeate O-methyltransferase (EC 2.1.1.68) . Enzyme activity and mRNA levels for both enzymes peaked in the middle of the growing season . These results strongly suggest that both forms of O-methyltransferase were actively participating in lignin precursor biosynthesis during the growing season . To determine the role of each enzyme form, xylem extracts from two days in the growing season were assayed with four substrates: caffeoyl-CoA, 5-hydroxyferuloyl-CoA, caffeate acid and 5-hydroxyferulic acid . Recombinant forms of caffeoyl-CoA and caffeate O-methyltransferase were also assayed with these substrates . The recombinant enzymes have different substrate specificity with the caffeoyl-CoA O-methyltransferase being essentially specific for CoA ester substrates with a preference for caffeoyl-CoA, while caffeate O-methyltransferase utilized all four substrates with a preference for the free acid forms . We suggest that caffeoyl-CoA O-methyltransferase is likely to be responsible for biosynthesis of lignin precursors in the guaiacyl pathway and may represent a more primitive enzyme form leftover from very early land plant evolution . Caffeate O-methyltransferase is more likely to be responsible for lignin precursor biosynthesis in the syringyl pathway, especially since it can catalyze methylation of 5-hydroxyferuloyl-CoA quite effectively . This latter enzyme form then may be considered a more recently evolved component of the lignin biosynthetic pathways of the evolutionarily advanced plants such as angiosperms. J Gen Virol, 1998 Sep, 79 ( Pt 9), 2275 - 81 cDNA cloning and molecular characterization of cherry green ring mottle virus; Zhang YP et al.; The complete nucleotide sequence of the cherry green ring mottle virus (CGRMV) genome was determined to be 8372 nt excluding a 3' poly(A) tail . Based on computer analysis and sequence comparison, five open reading frames (ORFs) were identified on the virion strand encoding: a putative RNA-dependent RNA polymerase, a triple gene block and a coat protein . Two other ORFs with Mr values over 10,000 and internal to the helicase and coat protein genes, but of unknown function, were also identified . Sequence and genome structure comparisons with other filamentous viruses indicated that CGRMV is most similar to apple stem pitting virus, some carlaviruses and potexviruses . However, it is different from members of any of